50% Effort. Molecular dissection of the pathway linking growth factor receptors to the nucleus: their role in normal cell growth and cancer. A synthetic biology approach to build, and hence understand, GPCR-regulated signaling networks in cancer: As part of our ongoing efforts addressing normal and aberrant cell growth control by GPCRs, we have begun using a synthetic biology approach consisting in the expression of mutant GPCRs that have lost the ability to respond to their natural ligands, but gained the ability to respond to a pharmacologically inert compound, clozapine-N-oxide (CNO). The use of these receptors activated solely by synthetic ligands (RASSLs) may enable us to build, and hence understand, GPCR-regulated signaling networks in normal and cancer cells. A large number of mitogens activate Gq-coupled GPCRs. Indeed, Gq-RASSL transduces potent mitogenic signals in NIH-3T3 cells, and is transforming if persistently activated, a process that requires the expression of c-Jun and c-Fos AP-1 family members. To investigate how GPCRs regulate AP-1-dependent gene transcription, we performed a genome-wide high-throughput RNAi screen in Drosophila S2 cells expressing Gq-coupled GPCR and an AP-1 reporter system. Both Jun and Fos, members of the AP1 dimer, were hits on our screen. We found that members of the Rho family of GTPases, specifically Rho and Rac, and their downstream effectors such as Pak and multiple regulators of Jun N-terminal kinase (JNK) were integral to AP-1 activation, while a JNK-specific phosphatase puckered, was a negative regulator. While molecules linking GPCRs to the hydrolysis of phosphatidylinositol and PKC activation were dispensable, we found that Trio, a Rho guanine nucleotide exchange factor (GEF) that binds directly to Gq, is essential for AP-1 activation by Gq-coupled GPCRs. Remarkably, Trio was found to be essential for the activation of JNK and p38 MAPKs, c-Jun and c-Fos expression, AP1 activation, and cell proliferation and transformation in mammalian cells. Collectively, the emerging information indicates that GPCRs are linked to the activation of AP-1 through a Rho-GTPase network that is governed by highly specific protein-protein interactions and phosphorylation events rather than by diffusible second messengers. GPCR-regulated signaling circuitries in cancer metastasis: Most tumor cells express numerous GPCRs that are activated by chemokines released to the tumor microenviroment by cancer and stromal cells, thus enhancing the motility and survival of tumor cells in an autocrine and paracrine fashion. In turn, cancer cells may gain, and thus can be selected for, the ability to co-opt the potent pro-migratory activity of chemokines and their GPCRs to metastasize to regional and distant organs. Among all GPCRs implicated in metastasis, we have initiated a focal effort on CXCR4, a GPCR that plays a critical role in many physiological processes involving cell migration, and its contribution to metastatic spread of some of the most prevalent human malignancies is well established. In a recently submitted study, we used multiple experimental strategies, including gene knock down, pharmacological tools, bioluminescence resonance energy transfer (BRET), videomicroscopy, and synthetic biology approaches, to show that in metastatic basal-like breast cancer cells, CXCR4-initiated motility and transendothelial migration strictly requires the activation of Rho through heterotrimeric G proteins of the G&#945;12/13 family. Furthermore, we provided evidence that interfering with the CXCR4-Rho signaling axis prevents the spontaneous metastasis of breast cancer cells, thus exposing new potential therapeutic targets for mechanism-based metastasis preventive strategies in breast cancer. 30% Effort. Molecular basis of developmental and tumor-induced angiogenesis. Molecular mechanisms by which Semaphorins and Plexins control angiogenesis: Semaphorin 3E (Sema3E) and its receptor Plexin-D1 control the patterning of the developing vasculature. However, little is known whether Sema3E-Plexin-D1 signals in adult and pathological angiogenesis. We have recently observed that Sema3E behaves as the most potent natural anti-angiogenic molecule in a number of in vivo models of developmental and tumor-induced angiogenesis. In particular, we observed that Sema3E provokes the rapid retraction of endothelial tip cells in the post-natal developing retinal vasculature, diminish the pro-angiogenic activity of VEGF in vitro and in vivo, and prevents the pro-angiogenic effect of head and neck cancer cells when grown in angioreactors in vivo. At the cellular level, Sema3E induces dramatic morphological changes in endothelial cells associated with the loss of integrin-mediated focal adhesive structures. Plexins are known to function as GTPase-activating proteins (GAPs) for R-Ras, which is required for repulsive responses to semaphorins in axon guidance. However, activation of Plexin-D1 by Sema3E induces cytoskeletal collapse without inactivating R-Ras. Furthermore, we found that Sema3E activates potently the small GTPase Arf6. Depletion of Arf6 by siRNA or expression of dominant negative mutants of Arf6 rescues Sema3E-induced focal adhesion disassembly. Emerging data suggest that the Sema3E-Plexin-D1 system negatively regulates angiogenesis by modulating integrin signal transduction via Arf6, thus revealing a novel anti-angiogenic molecular mechanism. 20% effort. AIDS-associated Kaposis sarcoma: molecular mechanisms. Development of alternative therapeutic approaches for KS management and prevention: Early work from our group led to the identification of the Akt/mTOR pathway as a critical signaling axis for KSHV-induced cancer progression, which contributed to the evaluation of rapamycin, an inhibitor of mTOR, for the treatment of KSHV-infected individuals who develop KS upon renal transplantation. As there is a possibility that the immunosuppressive effects of rapamycin may limit its therapeutic benefit, we have begun investigating the possibility of interfering with the Akt/mTOR pathway in an endothelial-specific manner without disturbing the critical function of mTOR in the immune system. We have recently found that vGPCR relies specifically on PI3K&#947;to initiate the activation of the Akt/mTOR pathway. PI3K&#947;, unlike the other PI3K isoforms &#945;, &#946;and &#948;, does not play a critical role in the immune system. RNA interference approaches suggest that PI3K&#947;is strictly required for vGPCR-induced tumorigenesis, and PI3K&#947;-specific inhibitors are as efficient as rapamycin in halting the development of vGPCR-induced tumors. We have now challenged these studies using genetically defined animal models. In particular, we used mice expressing Tva, a glycoprotein that acts as a receptor for avian retroviruses (ALV), in endothelial cells, thereby enabling the somatic expression of genes, including vGPCR, in vivo by cell-type specific retroviral gene delivery. When these mice were crossed with mice lacking PI3K&#947;, we observed that even littermates expressing a single allele of PI3K&#947;were partially resistant to vGPCR-induced tumorigenesis and death, and PI3K&#947;-deficient mice are completely protected. However, PI3K&#947;gene dosage had no effect on the lethality caused by infection with ALV-viruses expressing polyomavirus middle T antigen, which uses PI3K&#945;for its oncogenic activity. These studies may provide a rationale for the clinical evaluation of therapies exploiting the tissue-specific distribution of a critical component of the PI3K-mTOR pathway to treat KS in AIDS patients.